One mechanism by which human cancers become resistant to chemotherapy drugs is via overexpression of the human MDR1 gene. MDR1 encodes P170, a drug pump which prevents intracellular accumulation of cytotoxic drugs. Chemotherapy in combination with competitive inhibitors of P170 has therapeutic benefit in some types of cancer, but little is known about how P170 interacts with specific substrates. Naturally-occurring mutations in P170 alter substrate specificity, and studies with these mutants are beginning to elucidate the mechanism of P170-drug interaction. Additional information about how substrates interact with P170 will be applicable to designing new drugs and MDR1 inhibitors. The development of MDR1 genes conferring novel patterns of drug resistance will also be useful for gene therapy applications which use MDR1 as an in vivo selectable marker or chemoprotective agent. This proposal describes a genetic approach to understanding the mechanism of substrate utilization by P170. The specific aims are: 1) Clarify the mechanism by which P170 interacts with specific drugs by studying the effects of known mutations on P170 drug transport. Mutations known to alter substrate utilization by P170 will be engineered into MDR1 singly and in various combinations. Mutants will be tested for drug resistance and drug transport phenotypes. 2) Identify specific residues in P170 structural domains that are important for substrate interaction, using chimeric genes to map substrate binding determinants in ABC-transporters. Chimeras will be made between human P170 and structurally related transporters encoded by mouse mdr1 and yeast STE6 and STS1 genes. Transport properties of chimeras will be characterized using mammalian and yeast systems. 3) Further define the substrate binding domain(s) of P170 by using random mutagenesis to locate previously unidentified residues in P170 that are involved in substrate interaction. The long-term goals of these experiments are not only to define the structure of P170 as it relates to substrate utilization and transport, but also to enhance the ability to design rational chemotherapeutic and gene therapy approaches to the treatment of cancer.